Long term goals are to obtain a better understanding of cellular events involved in control of airway smooth muscle tone. We will study cholinergic mechanisms (a) at the level of the smooth muscle cell and (b) at the level of the paratracheal ganglia and myo-neural junction. The studies directed at coupling mechanisms in airway smooth muscle cells will determine if receptor-mediated activation of phosphoinositide metabolism generates signals which influence mechanisms by which receptor occupancy is coupled to contraction. These studies will require further development of methods for study and quantitation of phosphoinositide metabolism. We will attempt to inactivate phosphoinositide metabolism, as an approach to study relationships of messengers, generated by this system, and "coupling" mechanisms. We will determine if the putative phosphoinositide transducing system is influenced by second messengers generated by other cellular control mechanisms. We will study possible roles of phosphoinositide generated signals and airway muscle coupling mechanisms by use of phorbol esters, and determine effects of these agents on electrophysiological and contraction parameters. Experiments directed at the study of neurotransmission will determine basic properties of ganglion cells in the ferret trachealis muscle nerve-ganglion plexus. We will investigate mechanisms and function of after-hyperpolarizations, which follow action potentials in ganglion AH cells, and the nature and function of an apparent slow EPSP which is present in type B cells in this structure. We will develop and utilize new approaches to study (a) circuity of the ganglion plexus, and (b) coordination of inputs to smooth muscle cells. These include the use of multiple intracellular and extracellular electrodes, a fluoresent technique for visualizing single neurons under conditions where electrical measurements can be performed, and the use of intracapsular K electrodes. We will develop a preparation for studying electrophysiological properties of neurons in the guinea pig paratracheal ganglia, and to study neurons which provide non-adrenergic, non-cholinergic input to smooth muscle cells. These studies will give basic properties of two different steps involved in cholinergic activation of airway smooth muscle. They should set the groundwork for studies of airway muscle hypersensitivity using these approaches.